Summary: Project 3 Merkel cell polyomavirus (MCPyV)-positive Merkel cell carcinoma (MCC) presents a significant and timely opportunity to address basic questions in tumor immunology. Almost uniquely amongst human cancers, in virus-positive MCC (VP-MCC), a) a non-self, viral antigen is required for cell transformation, b) these oncoproteins are small and have few polymorphisms, c) the tumor mutation burden is very low, d) detection of viral oncoprotein-specific CD4 and CD8 T- and B-cells is very frequently possible, and e) biopsies are commonly obtained. Our studies of T-cell responses to the MCPyV T-antigen (T-Ag) provided the rationale for trials of anti-PD-1 therapy. These recently FDA-approved therapies have improved outcomes for many persons with advanced disease. Project 3 focuses on persons with local or regional disease, who collectively have a 37% chance of recurrence at 18 months after current standard therapy. Project 3 is led by an experienced viral immunologist and member with the P01 team. Our medical center is a well-established referral center for MCC, such that archived specimens from patients with known outcomes are available and 70-80 new patients are enrolled annually. To closely dissect the relationship between MCPyV-specific acquired immunity and outcomes, we propose three Aims. Aim 1 will determine the relationship between T-Ag-specific CD8 T-cell phenotype including dysfunction and avidity, with clinical outcomes, in persons with early-stage MCPyV (+) MCC. Aim 2 will conduct similar studies of the T-Ag-specific CD4 T-cells, including quantitative measures of tumor CD4 infiltration at the cell and molecular levels, and measurement of CD4 T helper phenotype. T-cell studies will focus on both TIL and blood, the site of both profound tumor-antigen specific T-cell localization and dysfunction. Results will be correlated with immunohistochemical studies of the suppressive tumor microenvironment. Aim 3 addresses the antibody response to T-Ag, which waxes and wanes with tumor burden in persons with MCPyV (+) MCC. Rising serum anti-T-Ag IgG presages tumor relapse and serial testing is included in 2018 NCCN guidelines for MCC care. T- Ag-specific B cells will be detected in blood using novel tetramer reagents and studied by IgG sequencing and detailed immunophenotyping. Our group has detected somatic hypermutation in the IgG genes of validated T- Ag-specific B-cells, indicating that these cells have traversed the germinal center, yet fail to differentiate into long-lived antibody secreting cells, a very unusual pattern. The underlying Premise of Project 3 is that insights we will deliver regarding adaptive immunity in VP-MCC will be generally applicable to malignancies that are harder to study because their tumor antigens are seldom conserved among patients.